UA81114C2 - Novel antibodies to tissue factor as anticoagulants - Google Patents

Abstract

This invention relates to the novel antibodies that bind with greater affinity to the factor VIIa/tissue factor (FVIIa/TF) complex than to tissue factor (TF) alone, do not compete for binding to TF with FVII and FX, an inhibit FX activation. The antibodies bind at the site of injury and prevent the initiation of thrombosis. The antibodies can be used to treat a variety of thrombotic conditions including but not limited to deep vein thrombosis, disseminated intravascular coagulation, and acute coronary syndrome.

Description

Description of the invention

For normal hemostasis, an important role is played by maintaining the necessary balance between procoagulation 2 and anticoagulation activity in blood vessels (|Oamie EMU., Viospetivighu, З0(43), 1991, pp. 10363-10370). A shift in the balance toward coagulation leads to thrombosis, which can lead to heart attack, "stroke," pulmonary embolism, and venous thrombosis. In connection with the above, there is a need for more effective and safer anticoagulants intended for the treatment of specific thrombosis-related diseases. 70 Tissue factor ("TF") is a transmembrane glycoprotein, which is the main initiator of the coagulation cascade | Metegvop U., Tpygotr. Naetozvi., 74(1), 1995, p. 180-184). Under normal physiological conditions, TF has no contact with blood. When a vessel is damaged, the entry of subendothelial TF and collagen into the blood leads to the activation of coagulation factors and platelets and, as a result, to the formation of a hemostatic plug.

TF, which has entered the blood, acts as a cofactor for factor Miia ("PMiiIa"), which catalyzes the activation of factor IX 12 CRIX") and factor X SEX"), which are crucial components of the body's tenase complexes and prothrombinase, respectively. This leads to the rapid formation of EC and thrombin. Thrombin then cleaves fibrinogen to form fibrin, which, in turn, polymerizes to form a fibrin clot. Inappropriate induction of TF expression in various clinical symptoms can lead to life-threatening thrombosis and/or cause pathological complications. The release of TF into the blood after plaque destruction is likely to lead to thrombotic occlusion, which is the cause of acute myocardial infarction and "stroke. In these cases, activation of proinflammatory signaling pathways by coagulation factors also leads to edema formation and infarct size.Vascular damage associated with vascular plastic surgery leads to upregulation of smooth muscle cell surface (SMO) TF, which presumably induces cellular signaling pathways associated with restenosis TF overexpression in tumor and Gram-negative sepsis leads to life-threatening thrombosis and activation of He) inflammatory pathways.

The EMIIIa/TF complex is involved in the pathogenesis of various thrombotic diseases, and the presence of a certain concentration of TF in the bloodstream is a risk factor for some types of patients. EMIP and TF play a unique role in the disruption of vascular function in the maintenance of hemostasis and the initiation of thrombosis in the event of vascular injury. WITH

TF is normally expressed in the adventitia, but in case of vascular disease there is an up-regulation and "- the normal expression of TF in the media and neointima is not characteristic of the organism. The expression of TF in atherosclerotic plaques increases, and upon contact with TF, blood protection carried out with the help of a thin fibrous membrane ("cap") may be disturbed. Surgical interventions, such as plastic surgery on vessels using "-- balloons, insertion of stents or endarterectomy, damage the vessel walls and lead to contact with TF, which

Zo is in deeper layers. Spontaneous destruction or endothelial. erosion of an atherosclerotic plaque rich in lipids leads to contact with TF and thrombosis, which causes unstable angina and myocardial infarction. TF can be in the bloodstream in microparticles of cellular origin, and the levels of TF in the bloodstream are increased in unstable angina, which suggests that TF, which is in the bloodstream, can participate in the formation of a thrombus. Sigstiayop, 99(22), 1999, pp. 2908-2913). Cancer is often associated with a state of hypercoagulation caused by overexpression of TF on the surface of tumor cells. This predisposes the patient to deep vein thrombosis, pulmonary embolism, etc

From" DVZ-syndrome (disseminated intravascular blood coagulation) of a low degree. DVZ leads to the deposition of fibrin in microvessels, which leads to disruption of many organs.

Anticoagulants based on proteins that target TF include TF-neutralizing antibodies that inhibit the 49 active site of factor Mia ("EMiIai"), tissue factor metabolic pathway inhibitor (TERI), and nematode anticoagulant protein ("MARS2"). Results obtained using models of thrombosis based on acute arterial injury, indicate that protein inhibitors of EMPa/TF are effective antithrombotic agents, which are characterized by a lower ability to cause bleeding compared to heparin, direct thrombin inhibitors, platelet inhibitors, and EXa inhibitors |Nitrbeg. . and - 70 others., Tygotr. Naetovzi. 85, 2001, pp. 475-481; Nagkkeg I.A. et al., Tygotr. Naetovzi. 74(1), 1995, pp. 464-472).

In addition, EMPA/7F inhibitors are more effective than other anticoagulants (eg, heparin,

T» EXa) regarding the ability to prevent neointima thickening and vascular stenosis after damage by an apa balloon

U. et al., Sigsshayop, 92(10), 1995, pp. 3041-30501.

With the help of experimental models of sepsis, it was established that the inhibition of TF, EMPA or the 22 EMIa/TtF complex is an effective antithrombotic way to prevent DVT and reduce mortality. Analogues of TERI

HF) prevent both thromboplastin-induced and endotoxin-induced DVT in rabbits |Oau K.S. etc., Viosd, 76, 1990, pp. 1538-1545); Vgedepdaga S. and others, Visod Soadii!. Ribgipoiuviv, 4, 1993, pp. 699-706). Monoclonal antibodies to EMIa |Vietopa V.). etc., Tygot. Naetosi, 73, 1993, pp. 223-230 or Iemi M. and others, 9. Siip.

Ipmevi., 93, 1994, p. 114-120) prevent endotoxin-induced DVZ in monkeys. Neutralizing TF antibodies, 60 EMIai and TERI inhibit DVZ and reduce mortality in baboons using the E. soy induced sepsis model | Steazeu A.A. etc., U. Siip. Ipmevi 91, 1993, pp. 2850-2860; Tauog B.V. etc., Viosd, 78, 1991a, pp. 364-368; Tauog R.V. etc., Site. ZposK, 33, 1991, pp. 127-134; Tauyug E.V. Naetoviaziv Zirri. 1 26, 1996, pp. 83-91). It is known that both EXa and the EMIa/TF/EXa complex induce the production of pro-inflammatory cytokines, which are associated with an increased risk of death in patients suffering from sepsis | Kiemaia M. et al., 62 Rgos. Mai), Asai. 5bi. ZA, 98, 2001, pp. 7742-7747). It is interesting to note that, as established using baboons as model animals, EMiIiai reduces the plasma levels of I/-6b and I/-8 | Gauyug B.V. et al., Viosa, 91, 1998, pp. 1609-1615), which suggests that inhibition of EMIa/TF may have an additional anti-inflammatory effect unrelated to other anticoagulation mechanisms.

Several antibodies are known from the literature that are effective anticoagulants and that bind to or neutralize either TF or the EMiIIa/TF complex, or both of them | see, for example, Sagzop 5.0. etc., Viosia, 66(1), 1985, pp. 152-156; Tapaka N. and others, Tygotr. Kevz., 40(6), 1985, pp. 745-756; Kigproiteg O. and others, Tygootr.

Naetozvi., 84(6), 2000, pp. 1072-1081; Kigsproieg O. and others, Viospetivighu, 40(3), 2001, pp. 675-682; Raeireg

Ki Yin., U. Moi. Biol., 313, 2001, pp. 83-87 and US patents 5506134, 5986065 and 6274142). Antibodies to 1TF proposed in this invention are effective anticoagulants that have improved characteristics compared to previously described antibodies to TF. In particular, the antibodies proposed in the invention bind with greater affinity to the EMIa/TF complex than to free TF, and they do not compete with EMII or EX for binding to TF.

The present invention relates to antibodies that act as anticoagulants that bind with greater affinity to the 7/5 MPa factor/tissue factor complex ("RMIPa/TF") than: to free tissue factor ("TF"). According to one of the embodiments of the invention, the antibodies proposed in the invention have at least a 2-fold higher affinity for binding to the EMiIa/TF complex than to free TF, as assessed by microcalorimetric analysis. According to a preferred embodiment of the invention, the antibodies proposed in the invention have at least a 5-fold higher affinity for binding to the complex

EMPalF than with free TF. According to an even more preferred embodiment of the invention, the antibodies proposed in the invention have at least a 10-fold higher affinity for binding to the complex

EMPalF than with free TF. According to another embodiment of the invention, the antibodies of the invention do not compete for TE binding with one or more coagulation factors selected from the group consisting of MI factors! (MIU, IIX (SRIHU) and X (EX). According to the preferred embodiment of the subject of the invention, the antibodies proposed in the invention do not compete for binding to TE with factors RMII and EX.

According to a more preferred embodiment of the invention, the antibodies proposed in the invention bind (8) with greater affinity to the EMIa/1F complex than to free TF and do not compete for binding to TF with factors

EMI and EH.

Anticoagulant antibody proposed in the invention carries out directed transfer and binds to "Ezo Complex of PMIa/TF in the area of damage and inhibits the activation of factor X (EX"), thereby preventing the formation of a thrombus and, as a result, is effective as an anticoagulant in the treatment of certain diseases, including (but not limited to) sepsis, disseminated intravascular coagulation, ischemic stroke, deep vein thrombosis, acute coronary syndrome, thrombosis-related complications after vascular plastic surgery, and coagulopathy in advanced cancer In addition, the antibody can be used in microvascular surgery, skin and vein transplantation, and organ transplantation.

Another object of the invention are pharmaceutical compositions containing the antibodies proposed in the invention.

The next object of the invention is a method of protecting the patient from the formation of a thrombus, which consists in the fact that the patient is administered a therapeutically effective amount of the antibody proposed in the invention, and thereby inhibits the formation of thrombin, while not having a direct effect on other coagulation parameters, « 470 such as platelet activation and aggregation. Another object of the invention is a method of reducing and treating deep vein thrombosis ("DVT") or disseminated intravascular coagulation ("DVT") or acute coronary syndrome or cancer with symptoms;" coagulopathy in a patient, which consists in the fact that the patient is administered a therapeutically effective amount of the antibody proposed in the invention.

The next object of the invention is a method of regulating the inflammatory response in a patient, which consists in the fact that

Go! the patient is administered a therapeutically effective amount of the antibody proposed in the invention.

Another object of the invention is the antibody proposed in the invention, which can be used to apply a non-thrombogenic coating to the surface of medical devices that come into contact with blood. - The next object of the invention is a set that contains the antibody proposed in the invention, which binds to the 5p complex of RMIPa/1F. In another embodiment, the kit may contain DNA sequences that encode antibody components. - The invention also proposes methods of obtaining the antibodies proposed in the invention, both by recombination and by synthesis.

The drawings show: in Fig. 1 - data on the activity of TF-binding single-chain antibodies, obtained on the basis of the analysis of the ability of pRTF/EMiIa to perform peptide hydrolysis (peptide hydrolysis). Analysis of the ability of RTF/EMIa to carry out peptide hydrolysis was carried out according to the method described in example 4, using a balanced

F) a mixture of EMIT (ZNM) and rTF (TONM), which is based on the affinity of EMIT to rTF (Coo(arru is "TONM). Hydrolysis of the chromogenic peptide substrate 52266 was evaluated according to the described method. The final concentrations of single-chain antibodies expressed in bacteria are presented and of the control proteins EMii (EMPa, which is inactivated by the chloromethyl ketone peptide, РРАСК) and MAt Mo4504 (Ategisap Oiadpowiis company); in Fig. 2 - data indicating that the binding of csEm(TE)Ze10 to rtF increases the apparent affinity of rtF to

Emiia. The analysis of the ability of rTP/EMiIa to carry out ceptide hydrolysis was carried out according to the method described in example 4, using 2NM of EMiia in the presence of ZVOONM expressed in bacteria with or without the antibody csEm(TE)Ze10. When performing this analysis, rTF was titrated and the rate of cleavage of the chromogenic b5 peptide substrate 52266 was determined. The apparent value of Ko for rTF was calculated using the standard approximation of the 4-parameter dependence;

in Fig. 3 - data on the activity of antibodies that bind TF, obtained by evaluating the prothrombin time (PT). The MP analysis was carried out according to the method described in example 4, using recombinant human thrombocytopenia (Yoade company, Ips.), containing full-length human TF in phospholipid vesicles. The final concentrations of single-chain antibodies expressed in bacteria and the control protein EMIai were determined; in Fig. 4 - data on the apparent affinity for binding csEm(TE)Ze10 to rTP. Analysis of the ability of rtF/EMPa to carry out peptide hydrolysis was performed according to the method described in example 4, using ZNM rtF and 2nM PMiia. The applied concentration of rTF was lower than the K p value, which characterizes binding with 7/0. Emia. Increasing concentrations of CsEm(TE)Ze10 expressed in bacteria were introduced, and the increasing reaction rate was used to determine the imaginary values of K p, which characterize the binding of this antibody to rTP, using the standard approximation of the 4-parameter dependence; Affinity of csEm(TE)Ze10 to the complex

RTF/RMIa (Co(arru"-b9nNM) was found to have a higher affinity than csEm(TE)Ze10 to RTF when measured using

VIAsoge (Co(drg)-47ONM); in Fig. 5 - data on the binding affinities of zsEm(TE)Ze10 with TF and the EMiIa/1F complex.

Microcalorimetric analysis was carried out according to the method described in example 4, using the antibody csEm(TE)Ze10, expressed in CHO cells. This analysis showed that zsEm(TE)Ze10 has - 20 times higher affinity for binding to the rTP/EMIa complex ("complex") than to rTP ("free TF"); in Fig. b6 - data on the dose-dependent ability of csEm(TE)Ze10 to inhibit the activation of EX. Analysis of activation of EX was carried out using the method described in example 4, using increasing concentrations of expressed in bacteria csEm(TE)Ze10, 250NM EX and the EMPa/T/F complex on the surface of phospholipids (10 pM EMPA).

The ISvo value represents the dose required to achieve 5095th inhibition; in Fig. 7 - data indicating that zsEm(TE)Ze10 non-competitively inhibits the activation of EX by the EMPA/TF complex. Analysis of EC activation was carried out according to the method described in example 4, using the bacterially expressed zsEM(TE)Ze10, 25-400 nm EC and the EMPa/TF complex on the surface of phospholipid (10 pM EMiIa). In this analysis, titrations were performed using increasing concentrations of i) csEM(TE)Ze10 (ONM, uncolored square; 0.25nM, uncolored diamond; 0.74nM, uncolored triangle; 2.2nM, uncolored circle; 6.7nM, colored diamond; 2OnNM, shaded triangle).

The presented graph of the dependence of I ipem"eameg-Vigk (1/5), where 5 (substrate) means factor X (μM); relative to 1/u, "g zo where M (speed) is expressed in the form of mOH/min., which characterizes hydrolysis of 52222 by ExXa, which takes place within 5 min.) indicates that cEm(TE)Ze10 is a non-competitive inhibitor with respect to the substrate, ie, X. All curves intersect on (or near) the abscissa, which is characteristic of non-competitive inhibition " - (in case of competitive inhibition, all curves should intersect on the ordinate axis); in Fig. 8 - data confirming the effectiveness of zsEM(TE)Ze10 on the model of ip mimo disseminated intravascular blood coagulation ("DVZ"). Antibody to TF zsEM( TE)Ze10 expressed in CHo cells was evaluated using the rat model of thromboembolism induced in Example 6 based on (A) percent mortality and (B) morbidity-mortality ratio.(A) For the vehicle-treated group the applied dose of TF caused 6095th mortality (I Ovo). Antibody with cEm(TE)Ze10 at a dose of 0.0 nmol/kg did not affect the mortality rate, but at a dose of 7.0 nmol/kg it reduced mortality to the level of "4095. (B) In the vehicle-treated ip mimo group, the applied TF dose resulted in an average morbidity-mortality rate of 2.6. Antibodies with CEM(TE)Ze10 at a dose of 0.7 nmol/kg did not affect mortality and had little or no effect on respiratory distress syndrome, but when used at a dose of 14 nmol/kg, it reduced the average morbidity-mortality index to " - 1.5.

Anticoagulant antibody proposed in this invention is an antibody that binds with greater affinity to the complex factor MIIIa/tissue factor (EMPa/TF") than to free tissue factor

Go! ("TF"). The antibody proposed in the invention has at least 2 times greater affinity, preferably with at least 5 times greater affinity and more preferably with at least 10 times greater affinity for binding to the EMiIa/TF complex than to free TF. In addition, the antibody of the invention does not compete for binding to the TF with one or more coagulation factors selected from the group that includes the MII factor

SEM), factor IX SRIH"Y) and factor X (EX). The preferred antibody proposed in the invention does not compete for binding with TF from EMII and with EX. i" Definition:

In this description, the following terms have the following meanings.

The term "recombinant proteins or polypeptides" refers to proteins or polypeptides obtained by means of recombinant DNA, that is, obtained from cells, microorganisms or mammals transformed with an exogenous DNA construct that encodes the desired polypeptide. Proteins or polypeptides expressed in the majority

F) bacterial cultures, must be free of glycan. Proteins or polypeptides expressed in yeast may have a glycosylation pattern different from the glycosylation pattern of proteins or polypeptides expressed in mammalian cells. The term "native" proteins or polypeptides refers to proteins or polypeptides isolated from a naturally occurring source. The term "native antibody" includes antibodies found in natural sources and their fragments.

The term "coding sequence" of DNA means a sequence of DNA, as a result of transcription of which mRNA is synthesized and which is translated into a polypeptide in the host cell, if it is located in such a way that it is under the control of the appropriate regulatory sequences. The boundaries of the coding sequence are defined by the start codon at the 5' M-end and the translation stop codon at the 3' C-end. The coding sequence may include prokaryotic sequences, cDNA derived from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, and synthetic DNA sequences. The transcription terminator sequence should, as a rule, be localized at the 3'-end relative to the coding sequence.

The term "nucleotide sequence" means a polymer of deoxyribonucleotides (bases adenine, guanine, thymine or cytosine). The DNA sequences encoding the antibodies of the present invention can be assembled from synthetic cDNA-derived DNA fragments and short oligonucleotide linkers to create a synthetic gene capable of being expressed in a recombinant expression vector. When discussing the structure of specific double-stranded DNA molecules, this description refers to the generally accepted designation of sequences only in the direction 5-53" of the non-transcribed strand of cDNA.

The term "recombinant expression vector" means a replication-competent DNA construct that is used either for amplification or for expression of DNA that encodes the antibodies of the present invention. The expression vector contains control sequences and a DNA coding sequence. Controlling DNA sequences include promoter sequences, ribosome binding sites, polyadenylation signals, 7/5 sequences of transcription terminators, upstream regulatory domains and enhancers.

The recombinant expression systems presented in the description should express antibodies after the induction of regulatory elements.

The term "transformed host cells" refers to cells transformed and transfected with exogenous

DNA. Exogenous DNA may not be integrated or may be integrated (covalently linked) into the chromosomal

DNA, complementing the genome of the cell. In prokaryotes and yeast, for example, exogenous DNA can be maintained on an episomal element such as a plasmid or be stably integrated into chromosomal DNA. As for eukaryotic cells, a stably transformed cell is a cell in which exogenous DNA is integrated into a replicative chromosome. Stability can be demonstrated by the ability of eukaryotic cell lines or clones to produce a population of daughter cells that contain exogenous DNA. with

The terms "analogue", "fragment", "derivative" and "variant" in relation to the antibodies proposed in the invention mean analogues, fragments, derivatives and variants of antibodies that retain substantially the same biological function or activity, and which will be more are described in detail below.

The term "analog" includes a propolypeptide, which includes the amino acid sequence of the antibody proposed in the invention. The active antibody of the invention can be cleaved from the additional amino acids that form the proantibody molecule by natural processes or by methods well known in the art, such as enzymatic or chemical cleavage. For example, - recombinant polypeptide csEM(TR)Ze10 (ZEO IYUO MO:1) is expressed in ! in the form of a propolypeptide, which "- consists of 282 amino acids, which is processed immediately with the release of an active mature polypeptide, which consists of 264 amino acids. -

The term "fragment" means a part of the antibody proposed in the invention, which retains substantially the same Ge) functional activity, which is found in the analyzes of IP miigo, described in more detail below.

The term "derivative" includes all modifications of the antibodies proposed in the invention, which mainly retain the necessary functions and carry an additional structure and have an accompanying function, for example, PEGylated antibodies, which have a longer half-life, and biotinylated antibodies, which will be further described below. Derivative " also includes M- or O-linked glycosylated antibodies, which can be obtained by embedding M-shshc or O-glycosylation sites in the antibody sequence using standard recombinant DNA techniques. The terms "substantially the same functional activity" and "substantially the same biological function or activity" each mean that the level of biological activity is about 30-10095 or more of the biological activity of the polypeptide being compared, if the biological the activity of each polypeptide is determined using the same method or analysis. For example, it can be said that an antibody has practically the same functional activity as the antibody from example 1 (ZEO IUO MO:1) if it, when evaluating peptide hydrolysis using rTP/ EMIa and EH activation using the assays described in Example 4 - demonstrates the ability to bind and neutralize the EMPa/1F complex - "Similarity" between two polypeptides is determined by comparing the amino acid sequence and Conservative amino acid substitutions of one polypeptide with the sequence of the second polypeptide. Such conservative substitutions include the substitutions described above in Te Ayaz oi Rgoivip Zedcepse apa Zygisishge 5 Bu

Ta» Bauyoii (1978) and in Agaoz EMVO.). 8, 1989, pp. 779-785). For example, amino acids that belong to one of the following groups are conservative substitutions: - AIa, Pgo, Su, Sip, Avp, Zeg, TAg; -Suv, Zeg, Tug, TAg; -Mai, Ne, Gei, Mei, Aa, RNe; (F, -Guv, Aga, Niv; ko -RNe, Tug, Tir, Niv; and -Avr, SIM. vo The term "antibody" in the context of this description includes intact molecules of immunoglobulins ("79"), as well as their fragments, such as Pa-, E(ar)"- and Em-fragments, which have the ability to bind to the epitope of the selected target protein, for example, soluble TF ("pTF"). As a rule, at least 6, 8 , 10 or 12 contiguous amino acids. However, epitopes that include non-contiguous amino acids may require a larger number, e.g., at least 15, 25 or 50 amino acids. 65 All other technical terms used herein have the meanings usually used by specialists in this field.

Antibodies proposed by the invention and their preparation:

Anticoagulation antibodies of the present invention bind with greater affinity to the MiIIa/tissue factor (EMIa/TF) complex than to free tissue factor ("TF"). According to a preferred embodiment of the invention, the antibodies of the invention , have at least a 2-fold greater affinity, more preferably at least a 5-fold greater affinity, and even more preferably at least a 10-fold greater affinity for binding to the EMiIa/TF complex than to free TF as assessed by microcalorimetric analysis. Accordingly in another preferred embodiment of the invention, the antibody also does not compete for binding to the TF with one or more coagulation factors selected from the group consisting of factor MI! "). According to the most preferred embodiment of the invention, the antibodies proposed in the invention do not compete for binding to TF, EM and EX.

According to the most preferred embodiment of the invention, the antibodies proposed in the invention bind with greater affinity to the EMIIa/TF complex than to free TF, and do not compete for binding to the TF from EMII and from EX.

In general, an antibody that specifically binds to a selected target protein (e.g., an EMPa//F or TF complex) when evaluated by an immunochemical assay gives a detection signal at least 5, 10, or 20 times higher than the detection signal other proteins. Preferably, antibodies that specifically bind to the selected target protein do not detect other proteins when evaluated by immunochemical assays and can perform immunoprecipitation of the target protein from solution.

The selected target protein can be used to immunize a mammal, such as a mouse, rabbit, guinea pig, monkey or human, to produce polyclonal antibodies. If necessary, the target protein can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and snail lymph hemocyanin. Depending on the host species, various adjuvants can be used to enhance the immunological response. Such adjuvants include (but are not limited to) Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surfactants (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, snail lymph hemocyanin, and dinitrophenol). Of the adjuvants used for humans, BCG (bacillus Calmette-Guérin) and Sogpurasiegisht ragmite are especially preferred.

Monoclonal antibodies that specifically bind to the selected target protein can be obtained using any method that provides the production of antibody molecules using stable cell lines in culture. These methods include (but are not limited to) the hybridoma-based method, the human B-cell hybridoma-based method, and the EVM-hybridoma-based method. Kopieg et al., May 256, 1985, pp. 495-497; -

Kogrog and others, y. And so on. Meiyodz 81, 1985, pp. 31-42; Soye et al. Rgos. May Asad All together. OBA 80, 1983, "- pp. 2026-2030; and Sogie et al., Moi. Sei| Axes 62, 1984, pp. 109-1201.

In addition, it is possible to apply the methods developed for obtaining "chimeric antibodies", splicing mouse -(Y7 antibody genes and human antibody genes to obtain a molecule with the appropriate antigenic specificity and biological activity |Moggizop et al., Rgos. Mai. Asad. Zsi OBA 81, 1984, pp. 6851-6855; Metsybregdeg and others,

Maighe 312, 1984, pp. 604-608; Takeda et al., Maishge 314, 1985, pp. 452-454). Monoclonal and other antibodies can also be "humanized" antibodies designed to prevent the patient from having an immune response to the antibody when it is used as a therapeutic agent. For direct application, the sequence of such "antibodies may have sufficient similarity to the sequence of human antibodies or may require changes of a few pt)s residues that are of crucial importance. Differences between rodent antibody sequences and human sequences can be minimized by substituting residues that differ from human residues;" sequences, by site-directed mutagenesis or by transplantation of complete hypervariable sites. In another embodiment, humanized antibodies can be obtained using the recombination methods described

In FshV821886388. Antibodies that specifically bind to the selected target protein may contain

Go! antigen-binding centers, which are either partially or fully humanized, (as described in Sh.5. 55653321.

In another variant, the techniques intended for the production of single-chain antibodies can be adapted using methods known in the field for the production of single-chain antibodies ("5cEU"), which specifically bind to the selected target protein. Antibodies with related specificity, but which have a different idiotypic 5p composition, can be obtained by shuffling chains from random combinatorial libraries of Id (|Vypop, Rgos. - Main. Asad. Zsi.O5A, 88, 1991, pp. 11120-11123). Single-chain antibodies can also be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template |TVigop et al., Etc. 9. Sapseg Rhem. 5, 1996, pp. 507-5111.

Single-chain antibodies can be mono- or bispecific and can be bivalent or trivalent. The construction of tetravalent bispecific single-chain antibodies is described, for example, in

Soota and Moghtizop, May). ViobesppoY 15, 1997, pp. 159-163). The construction of bivalent bispecific (F) single-chain antibodies is described in MaPepaag and Mozv, 9. VioYi. Spet., 269, 1994, pp. 199-2161. The nucleotide sequence that encodes a single-chain antibody can be constructed by manual or automated nucleotide synthesis, cloning into an expression construct using standard recombinant DNA techniques, and introduction into a cell for expression of the coding sequence. In another variant, single-chain antibodies can be obtained directly, using, for example, technology based on a filamentous veil display library |Megpaag et al., Ipi 9. Sapseg 61, 1995, pp. 497-501; and Misnoiv, etc., U. Ittipoi. May 165, 1993, pp. 81-911.

Antibodies that specifically bind to the selected target protein can also be obtained by inducing Ip bB UMO produced by the lymphocyte population or by screening Id libraries or panels of reagents that bind with high specificity, according to methods described in the literature (Opapayi et al. etc., Rhys State of Mai|.

Asai. 5 OBA 86, 1989, pp. 3833-3837; MMipieg et al., Mayte 349, 1991, pp. 293-299).

The DNA into which the antibody of the invention is cloned can be cloned in the form of cDNA or genomic DNA using any cloning procedure known to those skilled in the art (see, e.g., Zatbgook,

U.B. and others MoiIesshag Siopipd: A I arogaiyugu Mapiai, Soid Zrgipd Nagbrog I arogaiugu (1989)), publication incorporated in this description by reference).

In the event that the antibody is a monoclonal antibody, if a DNA sequence is identified that encodes an Eum fragment that, when expressed, has the ability to bind specifically, then antibodies that contain this Eum fragment can be obtained using methods well known to a person skilled in the art. Yes, 70 For example, in Spatsapag M.K. etc., Maishge, 339(6223), 1989, pp. 394-397; Weight 9.K. etc., 9. VioYii. Spent 265(25), 1990, pp. 15198-15202; Vaiga 9.K. etc., Rhos. May Asad All together. OBA 86(21), 1989, pp. 8545-8549;

Spatsanagu M.K. etc. Rgos. May Asad All together. OBA 87(3), 1990, pp. 1066-1070) (all publications are included in this description as a reference), the preparation of proteins of various single-chain antibodies is described.

According to the preferred variant, the TF-binding antibodies proposed in the invention are 7/5 single-chain antibodies obtained using a phage display library. The epitope-binding center of a single-chain antibody is constructed, which consists of two domains of the variable region: one from the heavy chain, and the other from the light chain. At the first stage of construction of the phage display library, genes of variable chains (Mn (with ZM), Mu and Mu) are cloned by PCR from nulled mRNA from human bone marrow, lymph node and spleen using a set of family-specific primers. The obtained libraries of pSITE-Mu (3.8x109 members), p7b04-MK (1.6x107) and p7604-M (3.2x107) are libraries of constant and having a wide variety of M-genes. Then My genes are amplified from the pRSITE-Mn library. With the help of PCR from the p26b04-M and p7604-M libraries, MK- and M, - genes that carry the reverse

Un region and a linker sequence at the 5-end. Then the gel-purified PCR products, which contain M n, MKki M, are ligated with each other, obtaining a set of csEm genes. A set of genes with cEm is cloned in the p2bO3 phagemid vector, and the product obtained as a result of lithiation is introduced by electroporation into competent cells of the E. soybean line o

TO1, creating a phage display library with sEm, labeled NiRPari Z, which carries 5.2 x 109 individual transformants (Kau V.K. et al. (1996) Rpade Oizriau oi Reriidev apa Rgoieipe: A I arogaiogu Mapiai, ed.

Asadetis Rgezz, Zap JuOiedo SA; Magke U.O. etc. 9U. My. Life 222(3), 1991, pp. 581-597; Zppeeiv M.O. etc.,

Rgos. May). Asad 5 OA 95(11), 1998, pp. 6157-61621. "AND

A TF-binding phage from a phage display library is selected, amplified and then identified using penning methods well known in the art. Soluble TF is immobilized on plastic tubes, and to reduce non-specific binding to plastic, you can use skim milk. A population of phage that expresses zsEm (zsEu-phage) is applied to the immobilized r/F in plastic tubes, and the unbound phage is removed by intensive washing. csEu-phage, which binds TF, is eluted from tubes and then amplified by infecting cells of E. soya TO1 in solution. The specified Penningui CO procedure is repeated three times, and the obtained cEmu-phage, which binds TF, is isolated by transforming cells of the TO1 line.

Transformants expressing antibodies that bind TF are identified using a standard method

EPIZA, for the implementation of which rTP is used, is immobilized on plastic 96-well plates. "

The single-chain antibody DNA inserts of EHIZA-positive transformants are sequenced. On the basis of sequencing, 6 unique single-chain antibodies were identified, i.e. cSem(TE)2c1, cSem(TP)2c11, cSem(TP)2a3, csSem(TE)2H6, cSem(TE)Ze10 and cSem(TE)ZH2, which express and are isolated from E. soy and the characteristics of which are described below in example 5. -» Expression and purification of antibodies proposed by the M invention:

There are several ways of expressing the recombinant antibodies proposed in the invention, including

E. soy, baculoviruses, yeast and mammalian cells or other expression systems. Methods of expression of cloned (ee) genes in bacteria are well known. To obtain a high level of expression of the cloned gene in the prokaryotic system, an important role is played by the construction of expression vectors that contain, at least, a strong promoter to ensure the termination of mRNA transcription. Examples of regulatory regions that can be used for this purpose are the promoter and operator region of the E. soybean beta-glucosidase gene, the genes of the tryptophan biosynthesis pathway - 20 BE. soybean, or the "reverse" promoter of lambda phage. It is possible to include selected markers in vector DNA, which are used to transform E. soybeans. Examples of such markers are genes that provide resistance to ampicillin,

I" of tetracycline or chloramphenicol.

For the expression of the antibodies proposed in the invention and their analogues, fragments, derivatives or variants, it is possible to use systems based on the cells of higher eukaryotic organisms, which can be selected from numerous

Cellular systems. Illustrative examples of mammalian cell lines include (but are not limited to) KRMI 7932, MEKO, and Nei! a, Chinese hamster ovary (CHO) cell lines, UMIZ8, VNK, СО5-7, С127, or MOSC. The predominant mammalian cell line is SNI -1. When the SNI-1 line is used, hygromycin is used as a selection marker for ko eukaryotic cells. SNI-1 cells are obtained from melanoma cells of the KRMI 7032 line, which is a readily available human cell line. Cell line SNI --1 in accordance with the Budapest Treaty 60 was deposited in ATCC on June 18, 1987. under the registration number SK. 9446. Cells that can be used according to the present invention can be obtained from ATCC. Illustrative examples of cells include cells of insects Zrodorieg Igidiregda and Votrbukh togi.

In a prokaryotic system such as E. soya, post/translational modification such as glycosylation cannot be carried out. In addition, proteins with a complex system of disulfide bridges often have assembly errors 65 when expressed in E. soybean. When using a prokaryotic system, the expressed protein is either present in the cell cytoplasm in an undissolved form in the form of so-called intracellular bodies, which are in the soluble fraction after cell lysis, or it is transferred to the periplasm by adding the appropriate secretory signal sequences. If the expressed protein is in insoluble intracellular bodies, solubilization and subsequent reassembly of the intracellular bodies is usually required.

Many commercially available prokaryotic expression vectors are known to those skilled in the art, such as pKK223-3 (Rpagtasia Ripe Spetisais, Uppsala, Sweden), pKK233-2 (Siopiesp, Palo Alto, CA, USA), and pOeMI (Firm of Rgoted Viojesi, Madison, Wisconsin, USA).

Promoters, which are usually used in recombinant expression systems based on microorganisms, include promoter systems of beta-lactamase (penicillinase) and lactose | Spapoa A.S. etc., Maishge, 275(5681), 7/0. 1978, pp. 617-624; Sboeade! OHM. et al., Mayige 281(5732), 1979, pp. 544-548), tryptophan (gr) promoter system (O.M. Soedayei et al., MysiI. Asidv Kev. 8(18), 1980, pp. 4057 -4074| and the promoter (as (|Mapiaii): T., etc.,

Moiesshiag Siopipd:. A Iarogaigu Mapiaii, So Zrgipd Nagrog Iarogaigu (1982) In other suitable bacterial expression systems, the phage lambda ri promoter and the heat-induced repressor of sii are used5857

IVegttaga N.O. and others Sepe, 5(1), 1979, pp. 59-76; Iome S.A. etc., Sepe 176(1-2), 1996, pp. 49-53).

Recombinant antibodies can also be expressed in yeast hosts such as Zasspagotuses segemisiae. This, as a rule, allows for various post-translational modifications. The expressed antibody can be secreted into the culture supernatant, which is free of many other protein residues, which simplifies purification. Yeast-based vectors designed to express the antibodies of the invention carry certain desired structures. Vector elements are typically derived from yeast and bacteria to enable the propagation of the plasmid in both cell types. Bacterial elements include a replication initiation site and a selectable marker. Yeast elements include the sequence of the replication initiation site (AK5), a selectable marker, a promoter, and a transcription terminator.

Suitable promoters in yeast-based expression vectors are promoters of the TKRI1 gene, the ASNI gene or

ARNI, acid phosphatase gene (RNOZ or RNOZ5), isocytochrome gene or promoters involved in glycolysis, such as the promoter of enolase, glyceraldehyde-3-phosphate dehydrogenase (ZAORN), 3-phosphoglycerate kinase (ROC), hexokinase, pyruvate kinase , triose phosphate isomerases and glucose phosphate isomerases |Niibetap K.A. etc., 9. Vioi. and)

Spent 255(24), 1980, pp. 12073-12080; Nezvv V. and others, U. Adm. Eplute Ked. 7, 1968, pp. 149-167; and NoYapa

M..). and NoPaga, 9.R., Viospetivhigu 17(23), 1978, pp. 4900-4907).

Commercially available yeast-based vectors include pUEZ2, pRISU (Impygodep, San Diego, CA), Wors-raONga, rusOE-1 (Uazpipowiup Kezeagsop, Seattle, WA), rVS102- K22 (ATSS

Mob7255) and UroOH265B5AI4 (ATSS Mob7233). Mammalian cell lines that can be used to express the recombinant antibodies proposed in the invention include (but are not limited to) CO5-7, I -cells, "-

C127, ZT3, Chinese hamster ovary cells (CHO), NeHa, VNK, SNI-1, M5O and NEK293. Recombinant proteins produced in mammalian cells are generally soluble and glycosidated and have authentic M-termini. --

Mammalian cell-based expression vectors may contain non-transcribed elements such as a replication initiation site, a promoter and enhancer, and 5- or 3- untranslated sequences, such as a ribosome binding site, a polyadenylation site, a splicing acceptor site and donor, and terminator sequences transcription.

Examples of promoters that are commonly used in mammalian cell-based expression vectors are viral promoters, such as promoters of polyoma virus, adenovirus, T-cell leukemia virus (TCL), simian "

Virus 40 (OV 40) and human cytomegalovirus (CMV). Depending on the selected expression system and the host, a homogeneous recombinant antibody can be obtained using various combinations of conventional chromatography methods used for protein purification. ;" These include: immunoaffinity chromatography, reverse phase chromatography, cation exchange chromatography, anion exchange chromatography, chromatography based on hydrophobic interactions,

Gel filtration and RHVR. If the expression system secretes the antibody into the nutrient medium, then the protein is possible

Go! clean directly from the media. If no antibody is secreted, it is isolated from cell lysates. Destruction of cells can be carried out using any generally accepted method, including - freeze-thaw cycles, ultrasound irradiation, mechanical destruction or the use of - cell-lysing agents.

For expression in bacteria of the single-chain antibody proposed in the invention, a plasmid construction based on РСАМТАВ5 (RPagtasia company) is used. For example, a plasmid containing a single-chain antibody zeEM(TE)Ze10 is a plasmid p2612/Ze10, and it encodes a sequence of a single-chain antibody, which is followed by the sequence of the e-yau tag used for protein purification. The amino acid sequence of the zsEM(TE)Ze10 antibody corresponds to 5BO IJU MO: 1 (example 1), and the DNA sequence encoding zsEm(TE)Ze10 5B corresponds to ZEO IJU MO:2.

For the plasmid construction, the basis of which is rINK5b25 | see 05 5827824), are used to express (F, single-chain antibodies proposed in this invention, in mammalian cells. For example, primers are designed to create a DNA fragment that overlaps the amino acid sequence of pro-zsEm(TE)Ze10, including the M-terminal signal sequence and the C-terminal e-iad sequence, in a bacterial expression plasmid PCR is performed to create a DNA fragment that is inserted into a mammalian expression plasmid containing the gene that confers resistance to ampicillin and the selection marker genes for hygromycin and ONEK ( dihydrofolate reductase).The expression of the single-chain antibodies proposed in the invention was under the control of the TKMRBOM promoter.

According to the preferred embodiment of the invention, the expression constructs intended for 65 mammalian cells are transfected by CHoO cells of the ОЧВ11 line. Select stable populations, using 40 Оmkg/ml hygromycin B in the environment НАМ5/Е12. Expression levels are approximately 50 Ωkg/l. To increase expression levels, 100NM methotrexate in alpha-MEM medium is used for population selection. The expression level of this population is approximately 5 mg/l.

Antibodies of the present invention can be purified using methods well known in the art. For example, antibodies can be purified using affinity chromatography on a column to which it is bound

TF The TF-antibody complex can then be eluted from the column using a high salt buffer.

The single-chain antibodies proposed in the invention contain the sequence e-iad at the C-end of the protein. Affinity chromatography columns containing antibodies to e-Iad were obtained from Ategisap/Rpagtasia Vioyesi. 70 Cell culture media were filtered through a 0.22-μm filter and applied to a 5-ml column loaded with e-Iad. at a rate of 2 ml/min. The column is washed with 0.2 M phosphate buffer supplemented with 0.0595 Mother, pH 7.0, and then the product is collected in tubes containing 0.1 volume of 1M Tris buffer, pH 2, for neutralization of elution buffer. In another variant, the filtered culture medium is applied to a column loaded with protein A. In this case, the column is washed with 50 mM citric acid, 00 mM Mass, 75 pH, 5 and eluted with the same buffer, pH 3.0. In both cases, the purified samples are then entered in the filled-in column

Zerpadeh 200, for separation of monomeric forms of antibodies from dimeric forms.

Selection of antibodies proposed in the invention:

After the antibodies are obtained, expressed and purified, they can be further characterized using

VIAsoge, analysis of evaluation of the rIF-dependent factor Miia (analysis of the ability of rIF/EMIC to carry out peptide hydrolysis), analysis of activation of EH and evaluation of PC, which are described in example 4, for the identification of antibodies proposed in this invention.

According to a preferred embodiment of the invention, csEm-antibodies that bind TF, which bind with greater affinity to the EMPa/TF complex than to free TF, are selected by analyzing the ability of pTF/EMiia to carry out peptide hydrolysis. When evaluated by this analysis, antibodies to TF, which bind with a higher affinity to the EMiia/TF complex than to free TF, should be characterized by higher values of K p(arru. Fig. 2 shows that for a single-chain antibody csEm( TE)Ze10 value of K du(arru o increases "5 times. Microcalorimetric analysis is used to evaluate the affinity of anti-TF antibodies to the EMIIIa/TF complex in comparison with one TF. Figure 5 shows that single-chain antibody csEm(TE)Ze10 is characterized by K values p binding to the EMiia/TF complex and to free TF, which constitute bONM and "I

ZZNM, respectively, which indicates a "20-fold increase in affinity to the EMIa/TF complex compared to free

TF Antibodies proposed in the present invention have at least 2-fold, preferably at least 5-fold (37 most preferably at least 10-fold) higher affinity to the EMIIIa/TF complex than to TF. "--

TvsEu-antibodies that bind TF that do not compete with EMI for binding to TF are selected by assaying the ability of RTF/EMiIIa to carry out peptide hydrolysis. In this assay, anti-TF antibodies that compete with EMIa for binding to TF should inhibit the hydrolysis of the chromogenic substrate 52266. Figure 1 shows that the single-chain antibody csEm(TE)Ze10 does not inhibit, but actually increases, EMIa activity. . ssEu-antibodies that bind TFs that inhibit activation of EX are selected using the evaluation of IF and the analysis of EX activation. When performing the analysis of AF, it should be expected that antibodies to TF, which prolong AF, should inhibit the activation of EX. Figure 3 shows that the single-chain antibody zsEm(TE)Ze10 prolongs the PM, which suggests that this antibody inhibits the activation of EX. In the analysis of EC activation, anti-TF antibodies that inhibit EC activity should inhibit the hydrolysis of the chromogenic substrate 52222. Fig.b shows that the single-chain antibody zsEM(TE)Ze10 also inhibits EC activity. "" csEu-antibodies that bind TF, which do not compete with EX for binding to TF, are selected using the EX activation assay. In this assay, anti-TF antibodies that do not compete with EX should inhibit activation

EX regardless of the applied concentration of EX. Figure 7 shows that the single-chain antibody csEm(TE)Ze1o inhibits EX activity regardless of the concentration of EX.

According to the preferred embodiment of the present invention, a single-chain antibody (csEm(TR)Ze10) was identified, which carries one M Ts/M,-binding center for TF. The amino acid sequence csEm(TE)Ze1o is presented in example 1 and corresponds to ZEO IU MO:1. The DNA sequence that encodes ssEm(TE)Ze10 corresponds to 5EC) - Analogues, fragments, derivatives and variants of the antibodies proposed in the invention:

And" By an analogue, fragment, derivative or variant of the antibodies proposed in this invention, we mean an analogue, fragment, derivative or variant in which: (I) one or more amino acid residues are replaced by a conservative or non-conservative amino acid residue (predominantly a conservative amino acid

Residue), and such a substituted amino acid residue may be encoded by the same or a different genetic code; or (II) in which one or more amino acid residues include a substituent group, or (I) in IF) in which the mature antibody is fused to another Compound, for example, a compound that increases the half-life of the antibody (eg, with polyethylene glycol), or ( IM) in which additional amino acids are fused to the mature antibody, such as a leader or secretory sequence or a sequence used to purify the mature antibody. From the given description, the concept of "analogues", "fragments", "derivatives" and "variants" should be obvious to specialists in this field.

Preferably, the derivatives proposed in this invention should contain conservative amino acid substitutions (further described below) of one or more predicted, preferably substituted amino acid residues. A "replaceable" amino acid residue is a residue that can be replaced in the sequence of the b5 protein of wild tyin without changing the biological activity, and an "indispensable" amino acid residue is necessary for the manifestation of biological activity. The term "conservative amino acid substitution" means a substitution in which an amino acid residue is replaced by an amino acid residue that has a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine , tryptophan, histidine). Non-conservative substitutions cannot relate to conserved amino acid residues 7/0 or amino acid residues located in the conserved domain of the protein, unless the substitutions are made for the purpose of selecting an antibody variant, which will be further described below. Fragments or biologically active proteins include polypeptide fragments suitable for use as a drug, research reagent, etc.

Fragments include peptides that contain amino acid sequences similar to or derived from the amino acid sequences of the antibody proposed in this invention, and which have at least one /5 the activity of the specified polypeptide, but which include fewer amino acids compared to the described full-length polypeptides.

In addition, preferred derivatives of the present invention include mature antibodies fused to another compound, e.g., a compound that prolongs the half-life of the polypeptide and/or reduces the potential immunogenicity of the polypeptide (e.g., polyethylene glycol, "PEGU"). PEG can be used to affect the 2o Water solubility, size, slow renal clearance rate and to reduce the immunogenicity of the antibody |see, for example, 05 6214966).In PEGylation, fusion of the antibody with PEG can be performed using any method known to those skilled in the art. For example, PEGylation can be carried out first by introducing cysteine by mutation into the antibody, followed by site-specific derivatization using PEG-maleimide.Cysteine can be added to the C-terminus of peptides. See, for example, Tzci8yti et al.

Rgos. May Asad All together. OBA 97(15), 2000, pp. 8548-85531). Another modification to which an antibody can be subjected includes biotinylation. In certain cases, the antibody may need to be biotinylated so that it readily i) reacts with streptavidin. Methods of protein biotinylation are well known in the art. In addition, it is possible to introduce an M- or O-glycosylation site in the antibody sequence so that post-translational M- or O-linked glycosylation of antibodies can occur immeasurably. Variants of antibodies proposed in this invention include polypeptides whose amino acid sequence is similar to the amino acid sequence of the original antibodies. The term "similar" means that the first amino acid sequence contains a sufficient or minimal number of identical or equivalent amino acid residues relative to the second amino acid sequence, so that the first and second amino acid sequences have a common structural domain and/or a common functional activity. For example, 17 amino acid sequences that contain a common structural domain that is identical for at least about 4595, preferably about 75-9895, are considered to be similar in the context of this description. Preferably, the variants should be similar to the amino acid sequence of the preferred antibodies proposed herein inventions

Variants include antibody variants encoded by a polynucleotide that hybridizes to a polynucleotide of the present invention or its complement under stringent conditions. Such variants, as a rule, "retain the functional activity of the antibodies proposed in this invention." Libraries can be used to create a heterogeneous population of fragments intended for screening and subsequent selection. fragments of polynucleotides. For example, a library of fragments can be created by processing и? of a double-stranded PCR fragment of a polynucleotide by a nuclease under conditions in which approximately only one single-strand break ("nick") per molecule occurs, denaturation of double-stranded DNA, renaturation of DNA with the formation of double-stranded DNA, which may include sense/antisense pairs from various resulting " nicks" of the products, removal of single-stranded sections from the transformed duplexes by treatment with nuclease 51 and incorporation by lithiation of the resulting library of fragments into the expression vector. With the help of this method, it is possible to create an expression library that encodes M-terminal and internal fragments of various sizes of the antibodies proposed in this invention.

Options include antibodies that have differences in the amino acid sequence as a result of mutagenesis. For example, mutagenesis can be carried out using recombinant DNA techniques, which are well known in this field, modifying the M, - and Mu regions of the Id light and heavy chains, creating variants that have a higher affinity for binding to the EMiIIa/ complex TF than with free TF. Particularly preferred options include antibodies that have modifications in the hypervariable regions of M, - and Mn regions.

Variants also include antibodies that have differences in amino acid sequence due to insertions or deletions of amino acid residues obtained as a result of mutagenesis. For example, mutagenesis can be carried out (F) using recombinant DNA techniques, which are well known in the art, to insert or delete amino acids in the M-terminal or C-terminal regions of M, - and Mu-regions of light and heavy chains of Id, yielding variants that retain virtually the same functional activity. Particularly preferred options include single-chain antibodies with insertions or deletions of amino acid residues in M H-Mi linkers between Mu- and

MiI plots. M D-M, -linker sequence in the single-chain antibody described in example 1 consists of 5 amino acid residues. Obviously, other short linker sequences consisting of 0-20 amino acids can be used if the antibody retains substantially similar functional activity.

Modifications of the existing MH-M, linker can be used to increase the stability of the dimeric form of the 65 single-chain antibody.

According to one embodiment of the invention, a library with a wide variety of variants is obtained by combinatorial mutagenesis at the nucleotide level, and it is encoded by a library with a wide variety of genes. A library with a wide variety of variants can be obtained, for example, by enzymatically ligating a mixture of synthetic oligonucleotides to gene sequences, such that a degenerate set of potential amino acid sequence variants can be expressed as individual polypeptides, or alternatively as larger fusion proteins (e.g. for phage display library), which contain a set of sequences. A number of methods are known that can be used to create libraries of potential variants from a degenerate oligonucleotide sequence. The chemical synthesis of the degenerate gene sequence can be carried out in an automatic DNA synthesizer and then 7/0 can be incorporated by ligating the synthetic gene into the appropriate expression vector. The use of a degenerate set of genes allows you to combine in one mixture all the sequences that encode the required set of sequences of potential variants. Methods of synthesis of degenerate oligonucleotides are known in this field | see, for example,

Magapo, Teiapedgop 39, 1983, p.3; Makiga and others, Appy. Chem. Viospet. 53, 1984a, p. 323; Makiga and others

Zsiepse 198, 19846, p. 1056; Ike and others, Mysivys Asii Kev. 11, 1983, p. 477).

Several methods are known in the art for screening the gene products of combinatorial libraries created by point mutations or truncation, and for screening cDNA libraries for the purpose of introducing gene products carrying a selected trait. Such methods can be adapted for rapid screening of gene libraries generated by combinatorial mutagenesis of antibodies for the ability to bind to TF- or to the complex

EMNa//F or the ability to inhibit the activation of EX. The most widely used methods that can be applied to large-scale screening of larger gene libraries generally involve cloning the gene library into replication-competent expression vectors, transforming the appropriate cells to yield the vector library, and expressing the combinatorial genes under conditions that facilitate detection of the required activity isolation of the vector encoding the gene whose product was detected. To identify the necessary variants, you can use recursive group mutagenesis (KEM), a method that ALLOWS to increase the frequency of functional mutants in libraries, in combination with screening methods.

Example 1 presents the amino acid sequence of one of the single-chain antibodies that bind ITP, (8) namely zsEm(TE)Ze10 (ZEO IYUO MO), and delinearized МН-М, linker and Mu- and Mi-sites. Variants that have the ability to bind TF or the EMI/TF complex or inhibit EX activation can be detected by screening combinatorial libraries of mutants, e.g., insertional, truncation, or point mutants of the antibodies proposed in the present invention, using an RTF assay. /EMIa to carry out peptide hydrolysis or EX activation analysis, which are described in example 4. Antibodies proposed in this invention include - antibodies described in examples 1 and 3 (ZEBEO OO MO:1 and 3), as well as antibodies that have insignificant variations "- sequences. The term "non-essential variation" includes any sequence variant associated with a substitution or deletion that substantially preserves at least one biological function of the antibodies proposed in this 77 invention, for example, the ability to inhibit the activation of EX. Such functional equivalents can preferably include antibodies in which the M domains or Mu regions are at least 9095 identical to the specified domains of a single-chain antibody having the sequence ZEO IU MO:1 or ZEO IO MOZ, and more preferably at least 95965 identical to domains M, - or Mu-sites of a single-chain antibody, which has the sequence ZEO IU MO:1 or 5EO IO MO:3, and even more preferably at least 9795 identical to the domains M,;- or Mp-sites " of a single-chain antibody, which has the sequence of ZEO IYUO MO: or 5EO IYUO MO:3, and also include areas in c of such an antibody, which have practically the same biological activity. However, the scope of this invention includes any antibody, the amino acid sequence of which has a slight variation relative to an antibody that has ZEO IU;" MO 1 or 5EO IJU MO:3, which is the functional equivalent, as will be described below.

Pharmaceutical compositions:

The invention also relates to pharmaceutical compositions that can be administered to a patient to achieve

Go! therapeutic action. The pharmaceutical compositions proposed in this invention can be prepared for administration by combining the antibody proposed in this invention, which has the required degree of purity and is taken in a pharmaceutically effective amount, with physiologically acceptable carriers. - Antibodies proposed in this invention can be used in the form of pharmaceutical compositions intended for intravenous administration or subcutaneous administration or intratracheal administration. - Thus, the antibodies described above are preferably combined with an acceptable sterile pharmaceutical carrier such as 595% dextrose, lactated Ringer's solution, normal saline, sterile water, or any other commercially available physiological buffer. intended for intravenous infusion. It should be apparent that the choice of carrier solution and dose, as well as the route of administration of the Composition, should vary depending on the patient and the specific clinical requirement and should be governed by standard medical procedures. (F) In accordance with the methods proposed in the present invention, the specified pharmaceutical compositions can be administered in amounts effective to inhibit pathological conditions associated with excessive thrombin generation in an individual. The antibody can be administered by bolus intravenous injection, by continuous intravenous infusion, or by combining both routes. In another or additional variant, the antibody, mixed with appropriate excipients, can enter the bloodstream from the intramuscular site. Systemic treatment with the antibody can be assessed by determining the activated partial thromboplastin time (APTU) on serial blood samples taken from the patient. The clotting time, which is determined in this experiment, 65 is prolonged when a sufficient antibody titer is reached in the bloodstream.

Recombinant antibodies and pharmaceutical compositions proposed in this invention can be used for parenteral, local, intravenous or local administration. Pharmaceutical compositions can be administered in the form of different standard doses of drugs depending on the method of administration. For example, standard dosage forms of drugs may be administered in forms including (but not limited to) tablets, capsules, powders, solutions, and emulsions.

Recombinant antibodies and pharmaceutical compositions proposed in this invention are most suitable for intravenous administration. Compositions intended for administration, as a rule, contain a solution of a single-chain antibody in a pharmaceutically acceptable carrier, preferably an aqueous carrier. You can use various aqueous media, for example, buffered physiological solution, etc. These solutions are sterile and generally free of 70 undesirable products. The compositions can be sterilized using conventional well-known sterilization methods.

A typical pharmaceutical composition for intravenous administration can be readily selected by one skilled in the art. The amounts that are administered are of course determined by the specificity of the protein and depend on its efficacy and pharmacokinetic profile. Modern methods of preparation of compositions that are administered parenterally are well known to specialists in this field and are described in more detail in Ketipdiop'z Rpagtaseciis! Zsiepse, 152 ed., ed

Mask Rybiizpipd Sotrapu, Easiop, Ra (1980).

Compositions containing the antibodies of the present invention or a mixture thereof (ie, a mixture with other proteins) can be administered as a therapeutic drug. For therapeutic use, the composition is administered to a patient suffering from a bleeding disorder or disease in an amount sufficient to treat or at least partially stop bleeding. The amount required for this is called the "therapeutically effective amount." The amount effective for this purpose should depend on the severity of the disease and the general health of the patient.

The compositions can be administered as a single dose or multiple doses depending on the dose level and required frequency of administration and tolerability by the patient. In any case, the composition should provide a sufficient amount of the proteins proposed in this invention to effectively treat the patient. i9)

The antibodies proposed in the invention, or their pharmaceutically acceptable compositions, are administered in a therapeutically effective amount, which should vary depending on various factors, such as the activity of the specific antibody used; metabolic stability and duration of action of the antibody; age, body weight, general state of health, sex and diet of the patient; way and time of introduction; output speed; a combination of medicines; the severity of a specific disease state; and the treatment to which the host is subjected. As a rule, the daily therapeutically effective amount is from about 0.14 to about 14.00 mg/kg body weight per day of the antibody proposed in the invention or its pharmaceutically acceptable composition; preferably from about 0.7 to about 1Omg/kg of body weight per day; and most preferably from about 1.4 to about 7.2 mg/kg body weight per day. For example, for administration to a human weighing 7Okg, doses should be from about 100mg to about 1.0g per day of an antibody of the invention or a pharmaceutically acceptable composition thereof, preferably from about 50 to about 7OOmg per day, and most preferably from about 100 to about

BOOmg per day. "

Cell and gene therapy:

The antibody proposed in the invention can be used in accordance with the present invention by expressing such an antibody ip mimo using a method called "cell therapy". For example, it is possible to create t cells that carry a polynucleotide (DNA or RNA) that encodes an antibody, and then inject the created t cells into a patient for treatment with the antibody. Such methods are well known in the art. For example, cells can be generated using procedures well known in the art by using a retroviral particle that contains RNA encoding an antibody of the present invention.(ee) An antibody of the invention can also be used in accordance with the present invention by expressing such an antibody ip mimo using a method called "gene therapy". So, for example, you can create - a virus that carries a polynucleotide (DNA or RNA) that codes for an antibody, and then inject the created virus into a patient - for treatment with an antibody. Such methods are well known in the art For example, recombinant adenoviruses that contain DNA encoding an antibody can be generated using procedures well known in the art proposed in this invention.

HT" Local introduction of anticoagulant antibodies proposed in this invention, using cell or gene therapy, can provide the introduction of a therapeutic agent into the target branch, that is, the lining of blood vessels, which consists of epithelial cells.

The scope of the invention includes both cellular and gene therapy methods. Several methods of transferring potentially therapeutic genes into certain cell populations are known (see, for example, iF) Miyidap, Zsiepse 260, 1993, pp. 926-931). These methods include: 1) direct transfer of genes | see, for example, UMoiy et al., Zsiepse, 247, 1990, pp. 1465-14681; 2) liposome-mediated DNA transfer | see, for example, Sariep et al., May 3, 1995, pp. 39-46; StuzviaI!, Maiige Mea. 1, 1995, pp. 15-17; bao and Nyapo, Viospet. Viorpuz. Ke5. Sott. 179, 1991, pp. 280-2851; 3) retrovirus-mediated DNA transfer | see, for example, Kau et al., Zsiepse 262, 1993, pp. 117-119; Apaeggop, 5siepse 256, 1992, pp. 808-8131; 4) DNA virus-mediated DNA transfer. Such DNA viruses include adenoviruses (predominantly Ad2- or Aa-based vectors), herpesviruses (predominantly herpes simplex-based vectors) and parvoviruses (predominantly vectors based on "defective or non-autonomous parvoviruses, more preferably adeno-associated virus-based vectors, more preferably vectors based on AAM-2) (See, e.g., AI et al., Sepe TNegaru 1, 1994, pp. 367-384; 5 4797368, incorporated herein by reference, and 51399411, incorporated herein by reference ).5 The choice of a particular vector system for the transfer of a gene of interest must depend on a number of factors. One important factor is the nature of the target cell population. Although retroviral vectors have been widely studied and found applications in various aspects of gene therapy, these vectors, such as generally cannot be used to infect non-dividing cells.In addition, retroviruses have potential carcinogenicity.

However, modern developments in the field of lentiviral vectors allow overcoming some of these limitations | see 70 Maiaipi et al., Zsiepse 272, 1996, pp. 263-2671.

Retroviruses from which the above retroviral plasmid vectors can be constructed include (but are not limited to) Moloney murine leukemia virus, splenic necrosis virus, retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, gibbon leukemia virus , human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and mammary tumor virus. In one embodiment of the invention, the retroviral plasmid vector is obtained from the Moloney murine leukemia virus.

The advantages of adenoviruses are their broad host range, their ability to infect resting or fully differentiated cells such as neurons or hepatocytes, and the fact that they are likely to be largely non-carcinogenic | see, for example, AM et al. (1994), above, p. 367)|. Adenoviruses probably do not integrate into the host genome. Because they exist outside the chromosomes, the risk of insertional mutagenesis is greatly reduced

IA and others (1994), above, p.37 31.

Adeno-associated viruses have the same advantages as adenovirus-based vectors. However, AAM is characterized by site-specific integration into human chromosome 19 |AII, etc. (1994), above, p. 3771.

According to a preferred embodiment of the invention, the DNA encoding the antibodies of the present invention is used in cell or gene therapy for disorders that include (but are not limited to) deep vein thrombosis, disseminated intravascular coagulation, acute coronary syndrome, or cancer with signs of coagulopathy.

According to this embodiment of the invention, cell or gene therapy using DNA that encodes the antibodies of the present invention is used to treat a patient in need thereof at diagnosis or immediately thereafter. It should be obvious to those skilled in the art that, according to this embodiment of the invention, any vector suitable for gene therapy can be used, which contains DNA that encodes an antibody - proposed in the invention or DNA that encodes analogs, fragments, derivatives or variants of the antibody "- proposed in the invention. Methods of constructing such a vector are well known | see, for example, Apdegzop MUR.

Mayte 392, 1998, pp. 25-30; Meghta I.M. and Botia M. Mayte 389, 1998, pp. 239-242)|. The introduction of the vector, which contains antibody DNA, into the target branch can be carried out by known methods. co

Vectors used for cell or gene therapy include one or more promoters.

Acceptable promoters that may be used include (but are not limited to) retrovirus TC; the OB-40 promoter and the human cytomegalovirus (CMV) promoter described in Mieg et al., Biochemistry 7(9), 1989, pp. 980-990), or any other promoter (eg, cellular promoters such as eukaryotic promoters cells, which include (but are not limited to) piston promoters, ro!PI, and B-actin). Other viral cs promoters that can be used include (but are not limited to) adenovirus promoters, thymidine kinase (TK) promoters, and parvovirus B19 promoters. The choice of a suitable promoter should be; will be apparent to those skilled in the art after reading this description.

The nucleotide sequence that encodes the antibody proposed in this invention is under the control of an acceptable promoter. Acceptable promoters that may be used include (but are not limited to)

Go! adenovirus promoters, such as the adenovirus major late promoter; or heterologous promoters, for example, cytomegalovirus (CMV) promoter; respiratory syncytial virus (RSV) promoter; - inducible promoters, for example, MMT promoter, metallothionein promoter; heat shock promoters; - albumin promoter; ApoA! promoter; human globin promoters; viral thymidine kinase promoters, 5p For example, herpes simplex virus thymidine kinase promoter; retroviral GTCs (including modified - retroviral AND TCs, which are described above); B-actin promoter; and human growth hormone promoter. Retroviral plasmid vector is used for transduction of packaging cell lines to obtain producer cell lines. Examples of packaging cells that can be transfected include (but are not limited to) REBO1, RAZ17, xr-2, y-AM, PA12, T19-14X; MT-19-17-N2, in SKE, usTIR, OR Ya-86, 5v OgRzhepuAt?i2 and SAM cell lines, which are described (in MiPeg, Nytap Sepe Tnegaru 1, 1990, pp. 5-14), the publication is fully included into this description by reference. Packaging cells can be transduced with the vector using iF) any methods known in the art. Such methods include (but are not limited to) electroporation, the use of liposomes, and precipitation with CaPoO). Alternatively, the retroviral plasmid vector can be encapsulated in a liposome or cross-linked to a lipid and then injected into the host. The producer cell line produces infectious retroviral particles that include the nucleotide sequence(s) encoding the polypeptide(s). Such particles of retroviral vectors can then be used to transduce eukaryotic cells with either ip migo or ip mimo. The transduced eukaryotic cells must express the nucleotide sequence(s) encoding the polypeptide(s).

Eukaryotic cells that can be transduced include (but are not limited to) embryonic stem b5 cells, embryonic cancer cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

An alternative approach to gene therapy is "transkaryotic therapy," in which a patient's cells are treated with ex mimo in order to induce silent chromosomal genes to produce the protein of interest after back-introduction into the patient's body. Transkaryotic therapy implies,

THAT the patient has the normal complement of genes required for activation. Transkaryotic therapy involves the introduction of a promoter or other exogenous regulatory sequence that has the ability to activate nascent genes into the chromosomal DNA of the patient's ex mimo cells, the cultivation and selection of cells that produce the active protein, and the subsequent reverse introduction of the activated cells into the patient's body in order for them to fully recover afterwards. After that, the "gene-activated" cell produces the protein of interest for a significant period of time, possibly throughout the patient's life. |O5 5641670 and 5733761) elaborate on this concept and are fully incorporated herein by reference.

Kits:

The present invention also relates to kits for research or diagnostic purposes. Kits typically include one or more containers that contain the antibodies of the present invention. According to a preferred embodiment of the invention, the kits include containers that contain single-chain antibodies in a form suitable for derivatization using a second molecule. According to the most preferred embodiment of the invention, the kits include containers that contain an antibody having the sequence ZEO IU

MO: 1 or 5EO IO MO:3.

According to another embodiment of the invention, the kits may contain DNA sequences that encode the 20 antibodies proposed in the invention. Preferably, the DNA sequences encoding these antibodies are present in a plasmid that can be used for transfection and expression in a host cell. The plasmid may contain a promoter (often an inducible promoter) to regulate DNA expression in the host cell. The plasmid may also contain appropriate restriction sites to facilitate the incorporation of other DNA sequences into the plasmid in order to obtain various antibodies. Plasmids can also contain numerous other elements designed to facilitate cloning and expression of encoded proteins. Such elements are well known to those skilled in the art and include, for example, selectable markers, initiation codons, termination codons, and the like. According to a more preferred (8) variant of the invention, the kits include containers that contain DNA sequences 5EO IU MO:2 or

ZEO and MO4.

Therapeutic indications: diseases in the etiology of which thrombus formation plays a very important role include myocardial infarction, disseminated intravascular coagulation, deep vein thrombosis, pulmonary embolism, ischemic "stroke", -- septic shock, acute respiratory distress syndrome, unstable angina pectoris and other arterial and venous conditions related to occlusion. Antibodies proposed in this invention can be used in all the indicated cases, as well as in other diseases in which the formation of a blood clot is pathological. Other pathological conditions in which the antibody proposed in this invention can be used include cancer, which is accompanied by coagulopathy and inflammation. Antibodies may also be used in skin and vein transplantation and organ transplantation. The concept of "can be used" ("useful") means that the antibodies are useful in treatment, or preventing the disease, or preventing its transition to a more serious stage. The antibodies proposed in this invention are also safe and effective anticoagulants, for example, they can be used in patients who have bioprostheses, for example, valves from the heart. These antibodies can replace heparin and warfarin in the treatment of, for example, pulmonary embolism or acute myocardial infarction. Antibodies proposed in this invention can also be used as;" coverage of medical devices where coagulation may occur.

Analyzes:

There are a number of laboratory tests that can be used to assess the effectiveness of anticoagulation

Go! activity of the antibody proposed in this invention. The anticoagulation effect of an antibody can be assessed based on plasma clotting time assays such as activated partial thromboplastin time (APT), thrombin time (thrombin-induced clotting time) ("PRT"), and/or prothrombin time ("PT"). These analyzes allow us to distinguish between different mechanisms of coagulation inhibition and include the activation of protein C. Prolongation of time

The folding detected in one of these assays suggests that the molecule can inhibit folding in plasma. The analyzes listed above are used to detect antibodies with anticoagulant activity that can bind TF both in purified systems and in plasma media. In addition, to evaluate the activity of the antibodies proposed in the invention, additional analyzes are used, such as the analysis of thrombin-catalyzed formation of fibrin from fibrinogen Shakiromueki N.M. etc., U. Vioi. Spent 261(8), two 1986, pp. 3876-3882)|, analysis of inhibition of factor M activation by thrombin (Evztop S.T. et al., 9. Vioi. Spet. 257(14), 1982, pp. 7944-7947 |), analysis of increased inhibition of thrombin by antithrombin I! heparin

F) cofactor I IEvtop M.Yi. etc., in Vioi Speth. 258(20), 1983, p.12238-122421), analysis of inhibition of thrombin activation of factor XII (RoYdag 9. et al., Tygotr. Naetovi. 58(1), 1987, p.140), analysis of inhibition of thrombin-mediated inactivation protein Z |Ppotrzop E.A. and Zaiet N.N., 9. Siip. Ipm 78(1), 1986, pp. 13-17) and analysis of inhibition of thrombin-mediated activation and aggregation of platelets (Eztop M... et al. (1983), above).

The following tests, described in detail in example 4, are used to evaluate the effectiveness of the drug or to evaluate the affinity of the drug to bind the antibodies proposed in the invention: 1) analysis of the ability of rTP/EMP to perform peptide hydrolysis; 2) analysis of factor X activation; 3) evaluation of the IF; and 4) microcalorimetric analysis. 65 In carrying out the methods used in the present invention, it should of course be apparent that references to specific buffers, media, reagents, cells, culture conditions, etc., should not be construed as

that " limit the scope of the invention, but as including all related products which, as will be apparent to those skilled in the art, may be of interest or significance in the particular context of the present disclosure. For example, one buffer system or culture medium may often be substituted for another , while achieving a similar or identical result.Those skilled in the art are aware of systems and techniques by which such substitutions can be made without undue experimentation and which can serve to optimize the methods and procedures presented herein.

Below, this invention will be described in more detail using non-limiting examples. It should be obvious that instead of the methods used in the specific examples, a specialist in this field certainly 7/0 can himself suggest other options for their implementation.

Above and in the following examples, unless otherwise indicated, all temperatures are given in degrees Celsius, all parts and percentages are presented in parts by mass. or in mass. 9o.

The complete description of all patent applications, patents and publications cited above is incorporated herein by reference.

Example 1

Construction of a single-chain antibody to TF zSem(TE)Ze1o (L)MISMIMISA AG ASIUMEREMAOMUMI KEZ 2 SSOSTtTIMOROSO5ZIKIVSAAZOBEZETOPAMUMZ M

Ukraine

Ani TPochuUumoMmMrUmsotmMtTM5АСО

SO5ZOSARMEMIETORNZMUMZAZROKTMUTISTTAE « " 58505MA5BUuMOMUOOOBROZBRTITMIHEOMN -

RO МРОКЕбЗО5ИО0Т5555А5171151КтТЕ0 --

Ed SOU OMIMMOBOTKI TtTMIAAA id g)

SsSARMRURYUORKERNKA A (264)

Single-chain anti-TF vzsEM(TE)Ze10 (ZEBO IYUO MO:1) consists of a signal peptide (from position -18 to position -1), M -section (1-126), M H-M,/-linker ( 127-131), M,-sites (132-246) and e-iad sequences (247-264). The DNA sequence zsEm(TE)Ze10 (5EO IU MO:2) encodes an amino acid sequence - with MO FAILURE. 1. And Example 2, "The design of a single -stranded antibodies to TF ZSM (Ti) ze10a -334 19) Misvmimimimimi Va and Assimmema Ce 5 (EE) And oostimorobziki5saazoesetamm M - U/" Kkkoaroke Ashimau 5I5I555511111111111111111111111111111111111 "UuUhUchsOoOMmMOLUuMOOoOtT MTU ASVS

SO5MEMGgTtTORNZUZAZROKTUT!ISTT55ZOo o 5vBMABUI MOM OOKROBZRITMIMEOMNKRB5 o SMROoOKE5O51I10T55M5A5VITIVvSIKTESLEAV and /IchXbO05UOMiMMBOOOTKkKITMIllLAAvAR

MRURORI ERA A (243)

The single-chain anti-TF zsEm(TE)Zet104 (ZEO IYUO MOO:Z3) consists of a signal peptide (from position -18 to position -1), M p-section (1-126), M H-M,/-linker ( 127-131) and M,-sites (132-243). 65 8sEM(TE)Ze10A differs from sSemM(TE)Ze10 by the deletion of three amino acids (ZAR) at the M-end of the MI domain and the deletion of the C-terminal e-iad signal sequence. The DNA sequence zsEm(TR)Ze10A (ZEO IU MO:4) encodes the amino acid sequence of ZEO IU MO:3.

Example C

Expression of anti-TF antibodies in bacterial and mammalian cells

Six different single-chain antibodies were identified from the TF-binding phage, csEzhm(TE)2c1,

ZSEM(TE)2c11, zSemM(TE)2az, zSem(Te)2p6, zSem(TE)Zet10 and zSem(TE)ZH2, which were overexpressed in E. soybean. and affinity purified using an e-iad affinity column as described above. The affinity of six purified antibodies to rTP was measured by ViAsoge, and these antibodies were then characterized in the rTP/EMIa peptide hydrolysis, EH activation, and microcalorimetric assays described in Example 4, the results of which are described in Example 5. csEM(TE) Ze10 antibody (5EO IU MO:1) was also expressed in CHO cells. The expression plasmid contained the DNA sequence encoding the csEm(TE)Ze10 antibody (ZEO IO MO:2), as well as hygromycin B and selected markers

ONEK Primary selection was carried out at 40 µg/ml hygromycin to select the population. The resulting population was then subjected to selection with TOOnNM methotrexate. In the course of this selection, cells that have /5 amplified copies of the DNA region containing the selected marker and the target gene are selected from the population. As a result of this selection, the expression levels were increased from about 0.Zmg/L to about bmg/L.

Example 4

Assays of IP migo efficiency and binding affinity. 1. Analysis of the ability of rTP/EMI to carry out peptide hydrolysis

The principle of this analysis is described below. The tripeptide para-nitroanilidamide bond of the substrate is hydrolyzed with the help of the rTP/EMIa complex. The release of the chromophore, that is, para-nitroanilide (p-MA), is evaluated at 405 nm and the concentration of the product formed per unit time is calculated using the molar extinction coefficient of 9920M "cm". The value of ISvo (C) is determined by approximating the initial velocities with help. of the 4-parameter equation М-(А-041(х/С)АВ)НО Га о

H-O-MaI-Hesh-Agd-r-MA -5H-O-Ma!-Hei-Agdjr-MA substrate 52266 tripeptide chromophore

Reagents and solutions: "g zo 1. Buffer for analysis (BA): 50mM Tris-HCl, 150mM Masi, 5mM Sasi", 0.195BSA, riH7.5 2. Human EMPA (NSMPA-0060, firm Naetaiiiodis Tesppoiodieev Ips.): 10-fold (x) working solution - 00/07 prepare a 20NM solution in buffer for analysis before use. "-

Z. Soluble TF (Vepeh): 10-fold working solution - prepare a ZONM solution in a buffer for analysis before use. - 4. Chromogenic substrate 52266 (Kari Rpagtasia Nerag Inc.): Stock solution: 10 mM in Na, which was stored at 490. 2.5-fold working solution - prepare a 2.58 M solution in the buffer for analysis before use. 5. Antibody: prepare 2.5-fold dilutions in buffer for analysis before use. "

Conditions for the analysis:

Analyzes are carried out in an O9b-well titration microplate at room temperature. The final concentrations of the components are: ;z» rtF ZNM

Antibody 1000-0.62508M

EUMa / gmn

Go! 82266. 1MM - Analysis procedure: - 1. Pipette 0.1ml of 2.5xBA (or control buffer) into each well. 2. Add 0.025 ml of 1OchRTF and incubate for 10 hours. at room temperature with gentle shaking. - 3. Add 0.025 ml of 10CHEMIIa, incubate for 10 hours. at room temperature with gentle shaking.

GT" 4. Add 0.1 ml of 2.5x substrate 52266, immediately transfer the tablet to the tablet reader and measure the kinetic characteristics of the enzyme at 405 nm for 15 minutes. with 10s intervals.

This analysis can be used to estimate the imaginary value of K p, which characterizes the binding of the antibodies proposed in the invention, to rIF or to the EMI/TF complex and to determine whether the antibodies proposed in the invention compete with EMI for binding to TF. i) 2. Analysis of activation of factor X ko The principle of this analysis is described below. EMIIa is incubated with amniotic fluid containing recombinant human

TF, obtaining a protease complex that has the ability to activate the substrate, i.e. EX. This complex is formed 60 in the presence (or absence) of various concentrations of antibody, then the substrate EX is introduced and the reactions are allowed to occur until the product is formed, that is, the active protease EX, which has the ability to hydrolyze the para-nitroanilide amide bond of the chromogenic substrate 52222. The release of the chromophore, i.e. of para-nitroanilide is evaluated at 405nm and the concentration of the product formed per unit time is calculated using the molar extinction coefficient of 9920M "cm". The value of IC 5o (C) is determined by approximating the initial velocities using the 4-parameter equation: M-(А-0О341ж(х/С) AB)О

B2-Pe-sty-c1Iu-Agd-r-MA - 582-Pe-sy-c1u-Agda-ONyar-MA substrate 52222 chromophore

Reagents and solutions: 1. Buffer for analysis: 50 M Tris-HCII, 150 mM Mass, 5 mM Sasi", 0.195 BSA, pH7.5. 2. Human EMITa (NSMIPA-0031, firm Naetaioiodis Tesppoiodiyev Ips.): 4-fold working solution - prepare a 100 pM solution in a buffer for analysis before use.

Z. Recombinant human TF (reconstructed in the applicant's laboratory from the preparation of Ippomip, Yuade): 70 working solution - prepare a dilution of 1:480 in the analysis buffer before use. 4. Human factor X (НСХ-0060, firm Naetayiodis Thesppoiodiez Ips.): 4-fold working solution - prepare a 1000nM solution in a buffer for analysis before use. 5. Chromogenic substrate 52222 (Kari Rpagtasia Nerag Ips.): Mother solution: bmM in HO, which was stored at 42C. Working solution - prepare a 0.78 mM solution in 3.57 mM EDTC (to stop the reaction), 150 mM mass, 50 mM Tris- NSI, pH7.5 before use. 6. Antibody:

Prepare 4-fold dilutions in assay buffer before use.

Conditions for the analysis:

Analyzes are carried out in a 9b-well titration microplate at room temperature. The final concentrations of the components are:

Bubbles rTF:1/4 from dilution 1:480

Antibody: 1000-0.6250M

EMna 2BpM

EX 2BONM c 82222 o., BabMM o

Analysis procedure: 1. Pipette 0.015 ml of 4xHBA (or control buffer) into each well. « 20 2. Add 0.015 ml of 4x blisters of RTF. 3. Add 0.015 ml of 4XhEMiIIa, incubate for 10 hours. at room temperature with gentle shaking. "- 4. Add 0.015 ml of 4XEX, incubate for 5 minutes. at room temperature with gentle shaking. "- 5. Add 0.14 ml of 52266, immediately transfer the tablet to the tablet reader and measure the kinetic characteristics of the enzyme at 405 nm for 15 minutes. with 10 with intervals. -

This assay can be used to determine whether the antibodies of the invention can co-inhibit the activation of EX by the EMIIa/TF complex and whether the antibodies of the invention can compete with EX for binding to the EMIPa/TF complex. 3. Determination of prothrombin time

When carrying out a standard reaction to determine the IF, 00 μl of the corresponding concentration of antibody or SFR "is added to the cuvette to a mixture containing 2 μl of thromboplastin IZ (Oade company) and 0 μl of 25 mM CaCl." The mixture is incubated for at 37 2C, then add 100 μl of citrated plasma (Neyepa I Arogaiyugiev company). In this other variant, an appropriate volume of concentrated antibody is added to 1000 μl of recombinant human "" thromboplastin (Ohio Kesotriazipi) and after approximately 2 minutes, 100 μl of reconstituted human plasma is added. The clotting time in each individual coagulation assay is estimated by determining the average of two of the measurements obtained using a device for measuring coagulation (coagulometer) (ee) tine Eiesiga 900C (Company Netoiapse) and determine the average values for the duplicated analyzes from (item-3 or 4). Dose-response curves for each inhibitor are obtained and then apply regression analysis to calculate the concentration (in NM) required to double the clotting time.- This assay can be used to assess the effect of the antibodies of the invention on the extrinsic tsu (uncharacterized) coagulation pathway. The amount of antibody required to doubling of the PF, which can be compared with other anticoagulants, recognized thus improving the relative efficacy of the antibodies proposed

Article" in the invention. 4. Microcalorimetric analysis

Microcalorimetric (isothermal titration calorimetry) analysis is used to determine the binding affinity (Kr) of the antibodies proposed in the invention to free TF or to the EMI/TF complex. This analysis is carried out using the device MisgoSa! MR-ITS. The EMIIIa/TF complex is preliminarily obtained by adding

Ф, 2.3-fold molar excess of EMIiIia to RTF. Gel filtration is used to confirm that rtTF in this analysis is completely included in the complex. To determine the affinity of the antibody to the complex, 1.2 µM MIa/TF complex is added to the cell of the microcalorimeter, and a 5 µM antibody solution is introduced into the syringe. To determine the affinity of antibodies to free rTP, 10 µM rTP is added to the cell and 141 µM antibody solution is introduced into the syringe. MisgoSai software is used for data analysis! Ogidip The data correspond to a single binding site.

Example 5

Characteristics of IP myo anti-TF antibodies proposed in the present invention 65 Six different TF-binding antibodies were isolated from a phage display library of complete human single-chain antibodies: zsem(TE)2c1, zsem(TE)2c11, zsemm(TP)2a3, cSEM(TR)216, cSem(TE)Ze10 and 5sEm(TE)ZN2.

The affinity of these antibodies, which bind rTF expressed in E. soya, when evaluated using ViAsoge, is 35-47O0NM. Analysis of the ability of pTP/PA to carry out peptide hydrolysis, which is described in example 4, is used to determine whether these antibodies can block the formation of an active MIPA/TP complex.

In this analysis, Miia binding to rTP increases the rate of cleavage of the chromogenic peptide substrate 52266 by more than 20 times. Antibodies that inhibit EMI binding to TF block this rate increase. For 5 single-chain antibodies, i.e. cSem(TE)2c1, cSem(TE)2c11, cSem(TP)243, cSem(TR)2y6 and cSem(TE)ZA2, the ability to inhibit the hydrolysis of 52266 was found, which suggests that they inhibit the The binding of EMI with RTF (Fig. 1). In contrast, the single-chain antibody csEm(TE)Ze10 does not inhibit 70 peptide hydrolysis, which is carried out by rTP/LPa, and in fact this antibody increases the rate of hydrolysis 52266, which suggests that csEm(TE)Ze10 increases the affinity of rIF to EMIa. Using the analysis of the ability of rtF/MIa to carry out peptide hydrolysis, it was established that the antibody zsEm("TE)Ze10 increases the affinity of EMpa to rtF, thus reducing the imaginary value of Kor by 5 times (Fig. 2). Antibody zsEm(TE)Ze10 does not affect the rate of hydrolysis carried out by EMI, in the absence of pRTF, indicating that this antibody does not interact with 7/5 Free EMI. The Kr value, which characterizes the binding of the antibody zsgzh(TE)Ze10 to rTP, determined by analyzing the ability of rIF/RMIPa to carry out peptide hydrolysis, is 654 nM (Fig. 4).

Microcalorimetric analysis is used to compare the affinity of csEm(TE)Ze10 to TF and to the complex

EmMPa/1F. These experiments allow us to establish that zsEm(TE)Ze10 expressed in mammalian cells has - 20 times higher affinity to the TF/EMiIa complex than to free rTF (ZZnm vs. bOOnm, Fig. 5).

Six single-chain TF-binding antibodies expressed in bacteria are compared using the EX activation assay and the IF assay. None of the five antibodies that inhibited the rate of hydrolysis of 52266 by the rTP/EMiIIa complex, namely cSem(TE)2c1, cSem(TE)2c11, cSem(TP)2a3, cSem(TP)216 and cSEzh(TE)ZN2 , do not prolong the AF compared to the control (SFR-buffer) (Fig. 3). In contrast, although the csEm(TE)Ze10 antibody does not have the highest affinity according to the data obtained with ViAsoge and increases the affinity of EMIa for rItF, the csEm(TE)Ze10 antibody is the only antibody in the group that inhibits the activation of EX ( Fig. b and data not presented) and prolongs the time of coagulation during the analysis of IF (Fig. 3). The IC 55 value, which characterizes the ability of the antibody i) zsEM(TE)Ze10 (dimer) to inhibit the activation of EX, is 0.44nM (Fig.b). Finally, an EX activation assay is used to determine whether the csEm(TE)Ze10 antibody can compete with EX for binding to the EMIIIa/TF complex. Antibody ssEzh(TE)Ze10 dose-dependently inhibits the activation of EX, while the value of Krd/(arr) «g zo Remains the same at all concentrations of the substrate EX, which indicates that zsEm(TE)Ze10 does not compete with

EX does not bind to TF or to the EMIa/1F complex in the same site as EX (Fig. 7). --

Antibody zsEm(TE)Ze10 was detected on the basis of binding to recombinant human soluble TF. The homology of the TF sequence between a human and a mouse or a human and a rabbit is 58 and 7195, respectively. Presumably, the antibody binds to a unique epitope on human TF, which affects the activation of EX by the EMPa/1/F complex. --

Physiologically, this antibody has an advantage over antibodies that compete with EMI or EMI for co-binding to TF. The value of K p, which characterizes the binding of EMI with soluble TF, is "1OHM (Fig. 2), this value is consistent with the published values for the binding of EMI with pTF (4.8 Nm, Metsepzspmapaeg R.R.

Her Moitizzeu ..N., U. Vioi. Spent 269(11), 1994, pp. 8007-8013 or for binding EMII, EMPA and inactivated with OIR. EMIiIai with full-size TF, reconstituted in neutral phosphatidylcholine vesicles |Vasp MK. and others, Viospetivhigu 25, 1986, pp. 4007-4020). The binding affinity of RMI! or EMIa with full-length TF increases significantly when the charged phosphotidyl serine is included in . phospholipid vesicles (Vasi K. et al. (1986), above), due to the interaction of the SI A-domain of EMII or EMIiA with charged i? membrane surface |Metsepzspu/lapdeg R.R. and Mogtizzeu U.N. (1994), above). Under these optimal conditions, EMIIIa binds to full-length TF with very high affinity (41 pM). In antibodies to TF, which competes for

Connecting with RMI! or ERMiIIa, the ability to compete for binding to this complex should be hindered

Go! EMPalF, which has a high affinity. In contrast, the csEm(TE)Ze10 antibody not only has a higher affinity to the EMIIIa/TF complex than to TF, but also does not compete with EMIIA for binding to TF. An antibody such as - csEM(TE)Ze10, which does not compete with EMI, should inhibit the activation of EX regardless of the concentration of PMII in plasma, which is -TONM.

Antibody zsEm(TE)Ze10 also has an advantage in comparison with antibodies that compete with EX for binding - with TF. The Ku value, which characterizes the binding of EH to the MIIIa/TF complex, is 0.061-0.099μM, which can be seen from the data presented in Fig. 7, this is consistent with the published values (O.μM, Vatsoi KM... and etc.,".

Axes Spent 275(37), 2000, pp. 28826-28833). The concentration of EH in human plasma is 140 nM (the K n value is 1.4-2 times higher). An antibody such as csEm(TE)Ze10, which does not compete with EX, which can be seen from Fig.7, should inhibit the activation of EX regardless of the concentration of EX in the plasma.

Example 6

F) The ip mimo thromboembolism model created in rats. The single-chain antibody csEm(TE)Ze1, which binds TF, is specific for primate TF. A model of thromboembolism caused by human TF (using as a reagent thromboplastin containing human recombinant TF, Ohio company) is created in conscious male Zrgadoe-Oauleu rats (350-400g, p»7/group). In this model of disseminated intravascular coagulation (DIC), TF induced by thromboplastin injection induces fibrin deposition in the lungs, thereby causing respiratory failure and death. Certain doses of mammalian cells expressing csEm(TE)Ze10 or the carrier are injected into the tail vein, and then after 15 min. perform a bolus injection of thromboplastin (0.5 ml/kg). In the group treated with the carrier, this dose of TF b5 causes the death of 6095 individuals (I Ovo), as a rule, after 5 min. injection of thromboplastin. The condition of rats is assessed using the following morbidity-mortality point system: 0 - absence of action; 1 - weak respiratory distress syndrome (recovery through ZOkhv.); 2 - serious respiratory distress syndrome (a state of agony, more breaths are needed for recovery); and Z - death. The average score is used to compare the effectiveness of the treatment of different groups. The results obtained in this IP MIMO analysis are presented in Fig.8. In this study, it was established that the antibody proposed in the invention has the ability to reduce mortality and reduce respiratory distress syndrome.

The examples described above can be reproduced, obtaining a similar result, by substituting in general or some of the described reactants and/or the test conditions used in the implementation of the above examples. 70 Although the invention is illustrated by the example of obtaining certain antibody constructs, it is obvious that variations and modifications may be made without departing from the spirit and scope of the invention.

Claims (24)

The formula of the invention, , , , 1. An anticoagulant antibody which is an antibody to tissue factor (TF) and which binds with greater affinity to the MiIa factor/tissue factor (TF) complex than to free tissue factor (TF) and which has the amino acid sequence represented in ZEO IU MO:1 or ZEO IU MO:3. 2. The antibody according to claim 1, where the antibody has at least a 2-fold higher affinity for binding to the EMIPal F complex than to free TF when measured by microcolorimetric analysis. C. The antibody of claim 2, wherein the antibody has at least 5 times higher affinity for binding to the EMIIIa/1TF complex than to free ITF. 4. The antibody according to item C, where the antibody has at least 10 times higher affinity for binding to the EMIIIa/1TF complex than to free ITF. high school 5. The antibody according to claim 1, where the antibody is a monoclonal antibody. 6. Antibody according to claim 5, where the antibody is a single-chain antibody, a Rar-dimer antibody or an antibody in (o) form dO. 7. Antibody according to claim 6, where the antibody is a single-chain antibody. 8. An antibody according to claim 7, wherein the antibody does not compete for binding to the TF with one or more Coagulation factors selected from the group that includes the MI factor! (RMII), factor IX (ХХ) and factor X (ЭХ). 9. Antibody according to claim 8, where the coagulation factors are EMII and EX. "- 10. The antibody of claim 1, wherein the antibody is glycosylated. "- 11. The antibody according to claim 1, where the antibody is modified by adding polyethylene glycol. 12. The antibody of claim 1, wherein the antibody is biotinylated to bind to streptavidin. - 13. An anticoagulant pharmaceutical composition containing an antibody according to claim 1, wherein the composition includes a pharmaceutically acceptable excipient and a therapeutically effective amount of the antibody. 14. A method of preventing thrombus formation, which consists in administering a therapeutically effective amount of the antibody of claim 1, wherein the antibody inhibits thrombin formation without directly affecting other parameters of coagulation, such as platelet activation and aggregation. "20 15. The method according to claim 14, where the method is designed to protect against thrombus formation in ischemic stroke, with thrombotic complications after plastic surgery on vessels or microvascular surgery. 16. A method of reducing and treating deep vein thrombosis (DVT), disseminated intravascular coagulation (DVT), acute coronary thrombosis or cancer with signs of coagulopathy in a patient, which consists in administering to the patient a therapeutically effective amount of the antibody according to claim 1 . 17. The method of regulating the inflammatory response in a patient, which consists in the fact that a therapeutically effective amount of the antibody according to claim 1 is administered to the patient. 18. The method of claim 17, wherein the inflammatory response is selected from the group consisting of sepsis, skin and vein grafts, and organ transplants. - 19. The antibody of claim 1, wherein the antibody can be used to form a non-thrombogenic coating on the surface of a medical device where the medical device is in contact with blood. - 20. Anticoagulation kit, which contains the antibody according to claim 1 and conventional excipients. Their" 21. An anticoagulation kit that contains DNA sequences encoding the antibody of claim 1 and conventional excipients. 22. A composition for gene therapy, which contains a polynucleotide sequence ZEO IU MO:2 or ZEO IUO MO4, which encodes an antibody, the amino acid sequence of which is represented in ZEO IU MO 1 or 5EO IUO MO:3, and this polynucleotide sequence is integrated into a suitable vector such in a way that is capable of expressing (F; proteins with amino acid sequences 5EO IYU MO:1 or ZEO IYUO MO:3. GI 23. An anticoagulation antibody that is an antibody to tissue factor (TF) and is a single-chain antibody that binds with greater affinity to the EMI/TF complex than to free TF, and where the antibody does not compete for binding to TF from RMII and EX, and which contains the amino acid sequence ZEO IU MO: 1 or 5EO IU MO:3. 24. A polynucleotide sequence that encodes an antibody according to claim 23, where the polynucleotide sequence contains the nucleotide sequence ZEO IU MO:2 or ZEO IU MO:4. b5